PROVIDED. Viruses and antiviral vaccines that enter the body through the skin or mucous membranes gain access to nearby lymph vessels and are transported to regional draining lymph nodes (LNs). When a virus arrives in a LN it must be prevented from continuing further along the lymph conduits, which would channel the pathogen into the systemic circulation causing viremia with potentially fatal consequences. LNs are believed to retain and neutralize lymph-borne micro-organisms, but the mechanisms are poorly understood. LNs also initiate adaptive immune responses to viral infections and vaccines because they recruit and harbor large numbers of lymphocytes and specialized antigenpresenting cells (APCs), which elicit protective effector responses, especially neutralizing antibodies. Our understanding of how lymph-borne viruses are presented to B cells is still very sketchy. Here, we prepose to use multi-photon intravital microscopy (MP-IVM) for time-and space-resolved visualization of the handling of lymph-borne fluorescent virions in LNs. We hope to obtain mechanistic insights into the cellular and molecular mechanisms by which LNs prevent pathogen dissemination and initiate adaptive humoral immunity. Based on preliminary work, our central hypothesis states that intranodal lymph conduits contain specialized cells that are highly adept at capturing viral particles and at presenting these particles to follicular B cells. This hypothesis will be tested in two specific aims: Aim 1 will explore the cellular mechanisms that determine the fate of lymph-borne viruses in LNs draining a subcutaneous injection site. Preliminary work has identified specialized macrophages (Mphs) in the floor of the subcaspular sinus (SCS) and the medulla of LNs that may function as a cellular 'flypaper'for viral particles and may be responsible for the filter activity of LNs. We will examine the phenotype and function of these virus-capturing Mphs (subaim 1.1) and address their role in the clearance and retention of different lymph-borne viruses (subaim 1.2). We will also investigate the origin and fate of LN-resident Mphs and other innate leukocytes and their relationship to a population of hematopoietic stem and progenitor cells that recirculate continuously between blood, tissues and lymph (subaim 1.3). Aim 2. will analyze where and how follicular B cells are exposed and respond to lymph-borne viruses. We will examine the sequential steps that are elicited by vesicular stomatitis virus (VSV) to induce early T-independent and late T-dependent humoral immunity in LNs. Subaim 2.1 will test the hypothesis that Mphs in the SCS function as early APCs for B cells. Subaim 2.2 will analyze T cell activation and B:T interactions as well as germinal center responses to viral challenge. Finally, subaim 2.3 will explore the possibility that polymeric nanoparticles could be devised to mimic invading viruses and provide a potent and versatile immuno-stimulatory platform for the delivery of future vaccines.